Ecological indicators for qualitative assessment of Ojarud River: A case study

Abstract Today, the application of ecological indicators based on organisms has replaced traditional saprobic approaches for assessment of the quality of rivers impaired due to organic pollution and some other environmental disturbances. This study aimed to weigh the quality of the Ojarud River in Ardabil, Iran, applying biological and physiological indices of macro‐invertebrates. A total of 12,524 samplings were fulfilled at four stations (S1, S2, S3, S4) from the headstream to downstream by a Surber sampler (30 × 30 cm2) from June/2020 to April/2021. All year round, the highest frequent families were Chironomidae (2658), Simuliidae (1025) from Diptera and Caenidae (1855), and Baetidae (724) from Ephemeroptera. The diversity pattern was analyzed by PAST software, and Primer 7 (BIO‐ENV analysis) was utilized to understand what factor has the most impact on the distribution of macro‐invertebrates. The least similarity of S4 to other stations was recognized by Cluster analysis. As per the ANOSIM (analysis of similarities), a statistically significant difference in the macroinvertebrates' frequency was established between S3 and other stations (p = .0001, r = .63). Moreover, the relationship between heavy metals and macro‐invertebrate showed that the three families of Simuliidae, Gomphidae, and Caenidae had a positive correlation with the concentrations of heavy metals in the sediment. As per the Ephemeroptera, Plecoptera and Trichoptera index, the water quality was placed in the “excellent” class, but the Biological Monitoring Working Party and Hilsenhoff Family Biotic Index indices scored the water quality “good” class at S1 and the “poor” class at S3. Based on the results of this study, the use of physicochemical and hydro‐morphological indicators can support the biological indicators but cannot replace them. In addition, careful evaluation of biological indicators is required to develop conservation strategies.


| INTRODUC TI ON
Out of the 17 Sustainable Development Goals (SDGs), "clean water and sanitation" is listed as the sixth goal of the United Nations (UN) goal to "Ensure availability and sustainable management of water and sanitation for all" (United Nations, 2022). As per the UN report, water-based ecosystems (mainly lakes and rivers) are swiftly being debased worldwide. Apart from the natural processes such as hydrological run-off, leaching from the soil, and rock weathering, anthropogenic activities have led to a vast discharge of industrial and domestic wastewater, and agricultural pollutants into natural waterbodies and intensively contributed to the poor water quality and loss of biodiversity of receiving aquatic environment.
Biological indicators have become an acceptable alternative to traditional approaches such as saprobic indices for assessment of the health status of river ecosystems (Ibáñez et al., 2010). The initial studies on applying biological indicators were carried out in Europe and the United States (Persoone & De Pauw, 1979). However, monitoring of river health via biological indicators becomes more prevalent in recent years in developing countries such as Iran (Aazami et al., 2015;Asadi Sharif et al., 2020;Shokri et al., 2014).

Fish species to detect habitat modifications and flow alterations
3. Macro-invertebrates to monitor the impact of acidification, hydro-morphological changes, and organic pollution on river health (Munyika et al., 2014;Rico-Sánchez et al., 2022).
The underlining reasons for the popularity of the macroinvertebrate-based bioassessment of river health could be its continuous monitoring of water quality in contrast to one-time sampling for chemical experiments, the abundance of taxa, their sedentary lifestyle and long lifetime, facial sampling and identification, presence in all areas of the river (crenal, rhithral, and potamal zones), distinct responses of each taxon to physicochemical pollutions, for example, acidification, eutrophication, and organic enrichment, and anthropogenic activities for example, river regulation, impoundment, and canalization, as well as playing a key role in effective river food web along with river continuum (Alba-Tercedor, 2006;Ficsór & Csabai, 2021;Sumudumali & Jayawardana, 2021).
The presence of heavy metals in water and/or sediment of rivers and their impacts on living organisms is a matter of serious concern.
Natural processes such as atmospheric precipitation, geological weathering, erosion, and bioturbation and/or anthropogenic activities, for example, industrial discharge, agricultural and urban activities mining, and transportation are among the main origins of heavy metals (Bradl, 2005). Unlike most pollutants, heavy metals are not bio-degradable and can be concentrated throughout the food chain and exert toxic effects on human health and the environment (Ali et al., 2019). Since macro-invertebrates engage in nutrient recycling and supply food to higher tropical levels, the increase in the concentration of heavy metals in rivers can lead to their accumulation to varying degrees in the food chain and disruption of its function.
The present study aimed to assess the health status of the Ojarud River in northwest of Iran. This river is not only the main source of water for agriculture in the area but also could offer opportunities to investigate some key factors as the main objectives of this study: (i) The effects of the anthropogenic stress on the macrobenthos diversity. It was especially important to know the effects of the sewage effluent, which was combined with a lot of foam in the third station ( Figure S1) on the distribution and diversity of the macrobenthic diversity of the studied river. (ii) The effect of the seasonal changes on the distribution of benthic invertebrates and also the replacement of pollutionsensitive species with pollution-resistant species. (iii) Validation of a number of well-performing biotic indexes (EPT, HFBI, and BMWP) and to know whether these biological indicators are effective to perform under the corresponding flora and fauna of the studied river. (iv) Assessment of the impact of heavy metals on benthic invertebrates as biological indicators. This has been given less attention in the biological monitoring of rivers as a strategic source of agricultural irrigation, and most studies only use biological indicators of benthic invertebrates for monitoring rivers.
As reported earlier, the combination of these two methods has provided more accurate and reliable results (Kaboré et al., 2022;Nahli et al., 2022). Accordingly, the study was designed to investigate the physicochemical aspects of the Ojarud River; identify its macro-invertebrates at the family level; assess the effects of the wastewater treatment plant as the point source of pollution on the health condition of the river; and apply PRIMER software to detect the most effective parameter on the spatial distribution of macro-invertebrates.

| Study region
Ojarud River, locally known as Germi Chai, located about 110 km from Ardabil City, Iran, originates from the Toulon River and drains into the Caspian Sea. The length of the Ojarud River is over 55 km and passes along a wastewater treatment plant where the ecosystem of this area is under physical and biological disturbances due to this source of pollution. Four sites were chosen along the longitudinal Germi Chai River gradient (illustrated in Figure 1) and samplings were carried from June 2020 to September 2021, starting in summer, and continuing in autumn, winter, and spring. The selection of sampling stations was per the pollution sources of the river (point and non-point): S1: The first station was selected at upstream of the river, where the aquatic plants grow nearby water all year round. S2: The second station was selected before the wastewater treatment plant. In this station also aquatic plants and freshwater algae were seen along the river.    preservation of the macro-invertebrates was carried out until the counting and laboratory analyses (APHA, 1999). The river water and sediment macro-invertebrates were identified at the family level using a stereo microscope (Oscoz et al., 2011).

| Sampling
The in-situ analysis of water's physical properties was carried out using a portable multi-parameter analyzer (WTW) to determine the pH, temperature (°C), dissolved oxygen (mg/L), and electrical conductivity (μs/cm). The phosphate and nitrate concentrations and biological oxygen demand (BOD) were determined using the American Public Health Association procedures (APHA, 1999).
An atomic absorption spectrophotometer (Varian SpectraAA 220 FS, USA) was employed to quantify the concentrations of heavy metals (cadmium and lead) in water, sediment, and selected macro-invertebrates.

| Biotic indices and diversity
Since the biological communities respond to environmental stress

| Data analysis
The statistical analysis of the data was performed using PAST sta-

| Macroinvertebrates
In the course of this project, 12,524 samples were collected and identified from four stations, which belonged to 10 orders and 17 families ( Table 2). The number of samples during the f seasons was highest in the order of spring (2974) > autumn (2444) > summer (2214) > winter (1892), respectively. Ephemeroptera and Diptera were the most frequent orders and Pulmonata, Lepidoptera, and Coleoptera were among the least frequent orders. As per seasonal data, the highest biomass of macro-invertebrates was recorded in summer (0.789 g/m 2 ) followed by spring (0.653 g/m 2 ), autumn (0.591 g/m 2 ), and winter (0.475 g/m 2 ). As per the stations, the highest biomass was observed in station 4 in summer

| Physical-chemical properties of the Ojarud River
The physicochemical properties of the river were determined in each season through both in-situ and ex-situ analyses. The data on the physical and chemical variables are given in Table 3. The maximum and minimum temperatures were, respectively, recorded in S3 (30.5°C) in summer and S1 (11°C) in winter. Although there was no statistically significant difference in temperature among the stations (p > .05), the inter-seasonal comparisons revealed a significant difference in temperature between winter with other seasons (p < .05). The maximum and minimum dissolved oxygen (DO) were, respectively, found in S1 (9.2 mg/L) in winter and S3 (7.6 mg/L) in autumn. In contrast, the highest concentration of biochemical oxygen demand (BOD) was recorded in S3 (18 mg/L) in summer, while it was the least in S1 (0.9 mg/L) in winter. As per the decreased water flow and increased point source pollution intensity, a rise in BOD was observed in S3 in summer. The concentration of NO − 3 varied between 63.278 mg/L (in S2 in winter) and 14.1 mg/L (in S1 in autumn). In contrast, the highest and lowest concentrations of PO 3− 4 were, respectively, recorded at S2 and S3 during the summer and spring (6.13 mg/L), and at S2 in autumn (0.25 mg/L).

| Heavy metals
To assess the heavy metal contamination status of the river, the concentrations of cadmium and lead were measured in water, sediment, and two macro-invertebrates in different seasons. In the upstream region, the average river cadmium was lower than that of the aquatic biological and surface water standards, while the concentration was significantly higher downstream of the river. In contrast, the concentration of lead in river water of S2, S3, and S4 was greater than that of the international standards. In contrast, the average concentrations of cadmium and lead in sediment were higher in the downstream stations than in the upstream region, however, it was still below the international standards of freshwater sediments (Figures 2 and 3).

| BIO-ENV analysis
Primer 7 (BIO-ENV analysis) was employed to understand what factor has the greatest impact on the distribution of macro-invertebrates.
The results of BIO-ENV statistical analysis showed that the most influential factors in the distribution of macro-invertebrates were physicochemical parameters (52%), sediment heavy metals (26%), and finally water heavy metals (10%), respectively (Figure 4). A comparison between water and sediment heavy metals showed that heavy metals in the sediment had a greater impact on the distribution of macro-invertebrates.

| Multivariate analysis and biotic indices
Analysis of similarities (ANOSIM) was used to compute the difference between the macro-invertebrates' communities among S1 to S4 based on Bray-Curtis dissimilarity index (Clarke, 1993). As per the results, S3 had a statistically significant difference with other stations in terms of the frequency of macro-invertebrate (p = .0001, r = .63; Figure 5). In addition, the SIMPER procedure (Clarke, 1993) was used to assess the average contribution of taxa to the dissimilarity (in percent) among the groups of the samples in four seasons. TA B L E 2 A list of macro-invertebrates recorded in four stations on the Ojarud River. The results of SIMPER analysis showed that the Chironomidae and Simuliidae families were the major taxa with the highest contribution to the differences among family groups. The community dissimilarity indices are indicated in Table 4.
The classical hierarchical cluster analysis based on a Bray-Curtis similarity index was used to compare the biomass similarity at four stations during all seasons. The results of this analysis revealed that S4 had the least similarity (23%) to other stations in three seasons (spring, summer, and autumn; Figure 6). Moreover, the relationships among the environmental factors, heavy metals, and biotic indices were assessed by canonical correspondence analysis (CCA). As per the CCA analysis, two biotic indices (BMWP,

HFBI) and biodiversity indices (Simpson and Shannon-Wiener)
showed a positive correlation with heavy metals. However, the EPT biotic index showed a positive correlation with temperature, dissolved oxygen, and pH and a negative correlation with heavy metals (Figure 7). On the contrary, the study on the relationship between heavy metals and macro-invertebrate revealed that three showed maximum values in S1 in autumn (1.9) while minimum values in S3 in summer (0.4). On the contrary, the Dominance diversity index (D) showed significant variations among the studied stations being highest in S3 in autumn (0.67) and lowest in S1 in winter (0.16).
The mean values of HFBI ranged from 4.53 to 7.56. The lowest value was at S1 in the winter and the highest value was at S3 in the winter (    (Odume et al., 2012;Rosa et al., 2014).

| DISCUSS ION
The results of the BIO-ENV analysis exposed the major influ-

F I G U R E 4
The output of BIO-ENV analysis.
the year. The results indicated that in the areas with moderate pollution and low altitude: the Diptera order, and in the areas with high altitudes, severe organic pollution, and suitable substrates (sand, silt derived from volcanic ash): the Oligochaeta order dominated. This was consistent with our results about the presence of the Diptera in pollution sites.
With reference to the CCA statistical analysis, among the biotic indicators, the EPT was positively correlated with TEM, DO, and pH and negatively with HM. Moreover, the EPT index showed a decreasing trend with increased organic pollution in S3 and an increasing trend with decreased pollution in S4. According to Wright and Ryan (2016), the EPT index was inadequate to measure the effects of heavy metals independently because some families (i.e., Hydropsychidae) in the taxa of the EPT index are resistant to toxic metals and various types of organic pollution. As reported, macroinvertebrates respond differently to heavy metals. However, sediments act as better heavy metal accumulators and serve as a more suitable predictor for metrics analysis (Ouma et al., 2022). Similar results were also observed in the present work wherein the sediment heavy metals exhibited higher effects on the distribution of macro-invertebrates in the studied sites. In contrast, the HFBI and BMWP biotic indices revealed the "good" class water quality for S1 and the "poor" class water quality for S3.
Literature is fraught with studies on the effect of point source pollution on water quality status (Asadi Sharif et al., 2020;Malvandi et al., 2021). In the study of Asadi-Sharif and Imanpur (Asadisharif & Imanpure, 2020), the effect of the point source pollution was assessed on the quality of the Disam River. The trend in diversity indices is presented in Figure 4. The Shannon-Wiener index is one of the most famous biodiversity indices, which was introduced in 1948 and comprised four classes based on the level of pollution and diversity (Shannon, 1948). The levels are as follows: (i) low diversity-high pollution, scored ≤1; (ii) low diversity-moderate pollution, scored 1 ≤ x ≤ 2; (iii) moderate diversity-low pollution, 2 ≤ x ≤ 3; (iv) high diversity-slight pollution,

| CON CLUS ION
The ecological quality of the Ojarud River, Ardabil, Iran, was as- The BIO-ENV statistical analysis showed that the physicochemical parameters (52%) were the most influential factors in the distribution of macro-invertebrates followed by sediment heavy metals (26%), and water heavy metals (10%). Besides, S4 showed the least similarity (23%) to other stations in three seasons (spring, summer, and autumn) as per the Bray-Curtis similarity index analysis. Above all, the water quality of the Ojarud River was scored in the "excellent" class based on the EPT index. Nonetheless, the BMWP and HFBI indices placed the water quality in the "good" class at S1 and the "poor" class at S3. The results of this study exposed a sizable alteration in the diversity and abundance of macro-invertebrate from the sensitive families to resistant and tolerant taxa under the contamination stress resulting from the point source pollution. The results of this study, yet again, verified the simultaneous applications of various indices for a more effective assessment of the health of a stream.

F I G U R E 7 Canonical correspondence analysis ordination diagrams of biotic indices and environmental factors in Ojarud
River.

F I G U R E 6
Cluster analysis of the macroinvertebrates' community collected in Ojarud River during four seasons in 2020-2021.

ACK N OWLED G M ENTS
The authors are grateful to the University of Mohaghegh Ardabili for the support provided. The support from the Ardabil University of Medical Sciences is also acknowledged.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data of the present study are archived in Dryad data repository and are publicly accessible. Dryad dataset doi: 10.5061/ dryad.18931zd2r.